Molecular detection of Trypanosoma species in sheep and goats in Mosul city

Molecular detection of Trypanosoma species in sheep and goats in Mosul city

Marwa Samir Mahmood and W.A. Alobaidii

Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

marwasamir958@gmail.com,

wasenamjad@yahoo.com, https://orcid.org/0000-0002-3090-9974

Abstract

In this study, we examined blood samples of 385 sheep and goats of different ages, sexes, and sources under routine microscopic examination of the blood smear (wet, thin, thick, buffy coat layer smears) to detect Trypanosoma. Results show that 81 samples were positive. These samples are succumbed to the molecular detection of Trypanosoma and other species by the extraction of parasitic DNA this parasitic DNA is detected in samples using KIN1, KIN2, and AITSF, AITSR primers. After that, conventional polymerase chain reaction was applied, and the results showed that 81 samples had a positive reaction in using KIN1 and KIN2 primers, while the positive samples were 76 when using AITSF, AITSR primers. Moreover, results showed a high rate of infection in sheep as compared with goats using both pairs of primers and two species of Trypanosoma in sheep and goats. Molecular was recorded, which include T. conglense and T. vivax. Animals more than 1-2 years old group showed a high rate of infection as compared with other ages group, and females have recorded a high rate of infection as compared with males. According to the source of animals, imported animals showed a high infective rate compared to native ones. This study is the first recorded Trypanosoma species in small ruminants in Mosul city.

 Keywords: Trypanosoma, T, conglense, T. vivax, Sheep, Goats

التقصی الجزیئی عن أنواع المثقبیات فی الضأن والماعز فی مدینة الموصل

مروة سمیر محمود و وسن امجد العبیدی

فرع الأحیاء المجهریة، کلیة الطب البیطری، جامعة الموصل، الموصل، العراق

الخلاصة

تم من خلال هذه الدراسة فحص عینات دم 385 رأساً من الضأن والماعز من مختلف الأعمار والأجناس والمصدر والتی تم فحصها تحت الفحص المجهری الروتینی من خلال فحص المسحات الدمویة (الرطبة، الخفیفة، السمیکة، مسحة طبقة الخلایا اللمفیة) وذلک للکشف عن طفیلی المثقبیات والتی أظهرت أن 81 عینة کانت موجبة، تم إخضاع هذه العینات الموجبة للکشف الجزیئی عن طفیلی المثقبیات والأنواع عن طریق استخلاص الحمض النووی الطفیلی ، ثم تم الکشف عن هذا الحمض النووی فی العینات باستخدام بادئات KIN1 و KIN2 و AITSF و AITSR ثم تم تطبیق تفاعل السلسلة المتبلمرة التقلیدی، وأظهرت النتائج أن 81 عینة أعطت تفاعلًا إیجابیًا باستخدام بادئات KIN1 و KIN2 بینما عند استخدام البادئات AITSF ، AITSR کان عدد العینات الموجبة 76. أظهرت النتائج إصابة عالیة فی الضأن عند مقارنتها بالماعز باستخدام زوج من البادئات فضلا عن انه تم الکشف الجزیئی عن نوعین من المثقبیات فی الأغنام والماعز T. conglense وT. vivax. أظهرت الحیوانات التی یزید عمرها عن 1-2 سنوات نسبة مرتفعة للإصابة عند مقارنتها مع الفئات العمریة الأخرى، وسجلت الإناث نسبة عالیة للإصابة عند مقارنتها بالذکور. وفقًا لمصدر الحیوانات، أظهرت الحیوانات المستوردة معدل إصابة مرتفعًا عند مقارنتها بالحیوانات المحلیة. وتعد هذه الدراسة هی الأولى من نوعها للکشف عن أنواع المثقبیات فی المجترات الصغیرة فی مدینة الموصل.

Introduction

Trypanosomosis is a protozoal microorganism caused by unicellular flagellated protozoa. It is classified with the Protozoa Sub-kingdom, Sarcomastigophora Phylum, Kinetoplastida Order, Trypanosomatidae Family, and Trypanosoma Genus: the genus Trypanosoma has two main groups: Stercoraria and Salivaria, which is found in blood, different body tissues, and fluids of the vertebrates (1). There are three subgenera of Trypanosoma that affect health: Vivax(Duttonella), congolense (Nannomonas), and Brucei (Trypanozoon) (2). Trypanosoma is transmitted by flies’ bites because the infective stages of the parasites are found in the mouth of the infected insect vector. Trypanosome’s species, namely: T. congolense, T. vivax, and T. brucei affects small and large ruminants (3). The intensity of the Trypanosoma infection depends on the animal’s spp, age of animals, and Trypanosomes spp, so the pathogenesis of Trypanosomosis differs according to the spp causing the disease (4). The main clinical signs are fever, anemia, loss of body condition, enlargement of lymph nodes, and abortion in pregnant animals. Diagnosis of Trypanosoma occurs by classical methods of microscopical examination of the blood smear (5). The microscopic examination does not detect the species and does not investigate multiple infections (6). The molecular method is sensitive and specific. The polymerase chain reaction (PCR) has pliable the amplification of specific DNA sequences. This method can be developed to detect many types of parasites. (7). The current study has been done because no molecular studies of detection the species of Trypanosoma in sheep and goats in Nineveh province.

Materials and methods

Animals and samples

Three hundred and eighty-five sheep and goats with different ages, sex, and source were examined under routine microscopic examination of the stained blood smear (wet, thin, thick and buffy coat layer smears) to detect of Trypanosoma. There were 81 positive samples. Then, molecular detection was done. Five ml of blood was collected from the jugular vein of each animal. The blood was transported to a tube which contained EDTA then stored in -20 ^o C.

DNA extraction and PCR protocol

DNA was extracted from the blood by using DNA blood extraction kit (Qiagen), the method of DNA extraction was done according to the manufacturer's instructions manuals. The first step of PCR was done to detect the Trypanosoma group. KIN primers universal Trypanosome can be used according to (8). PCR was performed in 25 ul volumes. The second step can be done according to Alex et al. (9) using AITSF and AITSR primers (Table 1). The amplification condition of PCR was done using a thermocycler (Table1). The final PCR products were separated using 1.5% agarose gel electrophoresis. Bands observed corresponded to T. congolense (Kilifi/Forest and Savannah); 650-800 bp, T. brucei; 520-540 bp, T. simiae; 440-500 bp, T. Godfrey; 320-400 bp, and T. vivax; 290-400 bp. (9). Figures 1 and 2.

Table 1: Primers and amplification condition which used in this study

Figure 1: Amplification of KIN1 and KIN2 primers, M=marker, 1-13 (650-700bp) positive to Trypanosoma, 14 Negative control.

Figure 2: Amplification of AITSF and AITSR primers, M=marker, 1,2,4,6-14 positive to T. conglense (700bp) 3,5 positive to T. vivax. (300-400bp) 15 negative control.

Results

Polymerase Chain Reaction results showed 100% of infection using Kin1 and Kin2 primers, while the percentage of infection was 93.8% using AITSF and AITSR primers in the same samples (Table 2). The PCR recorded a high infection rate in sheep and goats using both pairs of primers and AITSF, AITSR showed two species of Trypanosoma in sheep and goats (Table 3). According to age, the high prevalence rate of infection in sheep and goats is more than 1-2 years old, while the animals recorded a lower infection prevalence for more than two years (Table 4). Females of sheep and goats recorded high prevalence rate of infection with trypanosoma than males (Table 5). The high prevalence rate of infection with T. congolense in imported sheep and goats when compared with native once (Table 6).

Table 2: Showed infection with Trypanosoma using two pairs of primers

Table 3: Infection rate of Trypanosoma species in sheep and goats

Table 4: Results of infection of Trypanosoma species in sheep and goats according to age

Table 5: Results of infection rate of Trypanosoma species in sheep and goats according to gender

Table 6: Results of infection of Trypanosoma species in sheep and goats according to the source of animals

Discussion

Trypanosomiasis in animals is a significant problem to livestock development. Some rural areas of Africa affected by this disease. During the last few years, several researchers have detected many species in farm animals (10). In this study, the 81 samples detected using the routine microscopic method revealed that it could be positive using Kin1 and Kin2 primers, which can detect theTrypanosoma group, and 76 from 81 samples gave a positive result using AITSF and AITSR primers by the PCR technique. This primer recorded two species of Trypanosoma in sheep and goats with a high infective rate in sheep as compared with goats. Konnai et al. (11) reported that 18.3% percentage of infection in livestock of trypanosomes by using KIN primers and, he studied these primers' ability to investigate and differentiate the Trypanosoma using a single PCR (12). In this study, only two spp of Trypanosoma were recorded in sheep, including T. conglense and T. vivax, so T. conglense is predominant. Several studies take several spp of Trypanosoma in small ruminants (13) showed that T. vivax was the most dominant Trypanosoma in animals with an infection rate reached 20.91 %. T. simiae was rarely detected, only two goats and one sheep. At the same time, Bourzat and Gouteux (14) showed T. vivax with a high infection rate with clinical signs in animals, the increased prevalence of T. vivax approved by Authié et al. (15), which may result from the virulent of Trypanosoma, which is low and better controlled by animals, or from the mechanical transmission which has not recorded in the other spp, except T. congolense. The lower prevalence of T. congolense “forest type” when compared with T. vivax in domestic animals approved by Sidibe et al. (16); Bengaly et al., (17), which is due to the increased parasitemia in T. congolense with anemia which leads to the death of the animal. Another study showed that T. vivax, T. brucei, and T. congolense were absent in sheep but present in goats with low infection rates.

Franco et al. (18) and Daniel et al. (19) showed that the T. vivax, T. congolense, and T. brucei cause the infection in domestic sheep and goats, and T. vivax is dominant. The cause of mechanical transmission or decrease a time of development cycle in the tsetse-fly. Wayo et al. (20) showed that the most dominant spp of Trypanosomes was T. congolense in ruminants, and it approved the role of small ruminants as reservoirs of parasites. The reason for the difference between the results of the two pairs of primers could be the fact that sheep and goats may infect with other spp of Trypanosome (21), which records the infection of sheep with T. evansi.

Gael et al. (10) showed 5 Trypanosoma spp that infected goats (T. vivax, T. simiae, T. simiae T. savo, T. congolense, and T. brucei) and two spp that infected sheep (T. simiae and T. theileri) with increased trypanosome infection in both. Consequently, goats are more resistant to Trypanosoma than sheep, as Ng'ayo et al. (22) suggested. Sheep have a higher rate of infection when compared to goats and pigs. Findings show they are symmetrical with other studies that record that sheep are more infected than goats naturally (23). Another study showed that sheep were highly infected with T. congolense Kilifi and T. brucei, while pigs and goats were highly infected with T. vivax. (22), while Bedaso et al. (24) recorded 2 cases of T. vivax 1 sheep and one goat, rare of T. brucei, while Kiran and Idris (25) recorded a high prevalence rate among sheep was then the prevalence of the disease among goats.

Conclusions

Trypanosoma was affected by sheep and goats. T. conglense is more dominant than other species of these parasites in sheep and goats in Nineveh province.

Acknowledgment

The authors wish to thank the College of Veterinary Medicine, the University of Mosul for financially supporting this work, the veterinary teaching hospital for their support.

Conflict of interest

The authors declare no conflict of interest in the manuscript.